This project will elucidate the role of protein dynamics in enzyme function on all time scales, with the overall goal of developing enzyme design principles based on dynamics. We have pioneered new approaches to elucidate enzyme dynamics using structurally specific approaches, including isotope edited infrared spectroscopy coupled with ultrafast reaction initiation (T-jump or pH jump) and with fast microfluidics mixing methods. On the femtosecond time scale, we seek a deeper understanding of the fast atomic motions required to move the system over the transition barrier to achieve chemistry. On slower time scales (ps - ms) we seek to elucidate the conformational changes associated with substrate binding, catalytic site reorganization and product release. We focus on three enzymes, dihydrofolate reductase (DHFR) and in close collaboration with the Callender and Schramm labs respectively, lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP). The work is supported by theory and computation in the Schwartz group. The project has three specific aims: (1) Determine the conformational dynamics that control DHFR catalytic activity. This aim will test the hypothesis that the conformational dynamics of the Met20 loop act as a master control of DHFR activity by modulating the barriers to proton and hydride transfer. We will determine the effects of mutations discovered in our lab that perturb the H-bonding network on the proton transfer dynamics and any coupled protein dynamics, using pH jump methods and time-resolved IR spectroscopy. (2) Determine the protein structural dynamics that control the formation of the Michaelis complex in LDH. This aim will test the hypothesis that the Michaelis sub-state distribution and catalytic efficiency of LDH are controlled by the energy landscape of the catalytically important loop motions. We plan to determine how these loop motions are related to the sub-state distribution and the extent to which the protein conformational distribution is collapsed in the observed sub-states using ultrafast mixing, coupled with T-jump experiments in the Callender lab. Calculations by the Schwartz group will identify the dominant Michaelis configurations and measure the dynamics of transitions between them. These calculations will enable the interpretation of the dynamics observed in our experiments. (3) Investigate the relationships between protein structural dynamics, pathways for energy flow and allostery in enzymes. This aim will test the hypothesis that allostery requires pathways for energy flow to reach a specific target that depend on the protein structure and its dynamics. We have developed ultrafast, pump-probe IR spectroscopy to probe the specific pathways of energy flow in enzymes. We will apply these methods to characterize the dynamics of energy flow in DHFR, LDH and PNP, and how it depends on inhibitor binding and the distribution of the conformational sub-states of the enzymes. We expect tight binding dynamic inhibitors to have very different energy flow dynamics than less efficient inhibitors that cause conformational collapse.